Cells usually respond to changing growth conditions with a change in the specific growth rate (μ) and adjustment of their proteome to adapt and maintain metabolic efficiency. Description of the principles behind proteome resource allocation is important for understanding metabolic regulation in response to changing μ. Thus, we analysed the proteome resource allocation dynamics of Escherichia coli into different metabolic processes in response to changing μ. E. coli was grown on minimal and defined rich media in steady state continuous cultures at different μ and characterised combining two LC-MS/MS-based proteomics methods: stable isotope labelling by amino acids in cell culture (SILAC) and intensity based label-free absolute quantification. We detected slowly growing cells investing more proteome resources in energy generation and carbohydrate transport and metabolism whereas for achieving faster growth cells needed to devote most resources to translation and processes closely related to the protein synthesis pipeline. Furthermore, down-regulation of energy generation and carbohydrate metabolism proteins with faster growth displayed very similar expression dynamics with the global transcriptional regulator CRP (cyclic AMP receptor protein), pointing to a dominant protein resource allocating role of this protein. Our data also suggest that acetate overflow may be the result of global proteome resource optimisation as cells saved proteome resources by switching from fully respiratory to respiro-fermentative growth. The presented results give a quantitative overview of how E. coli adjusts its proteome to achieve faster growth and in future could contribute to the design of more efficient cell factories through proteome optimisation.

Depending on the environmental conditions, cells adapt their metabolism and specific growth rate. Rearrangements occur on many different levels such as macromolecular composition, gene and protein expression, morphology and metabolic flux patterns. As the interplay of these processes also determines the output of a recombinant protein producing system, having control over specific growth rate of the culture is advantageous. Continuous culture methods were developed to grow cells in a constant environment and have been used for decades to study basic microbial physiology in a controlled and reproducible manner. Our review summarizes the uses of continuous cultures in cell physiology studies and process development, with a focus on recombinant protein-producing microorganisms.

Bacteria are widely used as hosts for recombinant protein production due to their rapid growth, simple media requirement and ability to produce high yields of correctly folded proteins. Overproduction of recombinant proteins may impose metabolic burden to host cells, triggering various stress responses, and the ability of the cells to cope with such stresses is an important factor affecting both cell growth and product yield. Here, we present a versatile plasmid-based reporter system for efficient analysis of metabolic responses associated with availability of cellular resources utilized for recombinant protein production and host capacity to synthesize correctly folded proteins. The reporter plasmid is based on the broad-host range RK2 minimal replicon and harbors the strong and inducible XylS/Pm regulator/promoter system, the ppGpp-regulated ribosomal protein promoter PrpsJ, and the σ32-dependent synthetic tandem promoter Pibpfxs, each controlling expression of one distinguishable fluorescent protein. We characterized the responsiveness of all three reporters in Escherichia coli by quantitative fluorescence measurements in cell cultures cultivated under different growth and stress conditions. We also validated the broad-host range application potential of the reporter plasmid by using Pseudomonas putida and Azotobacter vinelandii as hosts. The plasmid-based reporter system can be used for analysis of the total inducible recombinant protein production, the translational capacity measured as transcription level of ribosomal protein genes and the heat shock-like response revealing aberrant protein folding in all studied Gram-negative bacterial strains.